A replication-competent Ad4-HIV recombinant encoding a mosaic gag gene is being produced under cGMP conditions for use in a phase I clinical trial. This clinical grade vaccine will be evaluated following oral administration for safety, initial immunogenicity, and dose level. Additionally, this product will enable comparative immunognicity studies with other vectored HIV vaccines to facilitate selection of candidate vaccines to move forward to phase II and eventually phase III testing. We are also developing an Ad4-HIVenv recombinant. Eventual production of this candidate vaccine will allow testing of the replication-competent Ad-recombinant prime/envelope protein boost approach which has elicited such potent protective efficacy in pre-clinical non-human primate studies. In additional pre-clinical studies, Ad recombinants containing the green fluorescent protein (GFP) have been generated and are being used in studies in vitro for optimization of transfection procedures. In addition, use of these GFP recombinants in vivo has allowed biodistribution studies of the Ad-recombinant in the rhesus macaque model following administration by several different mucosal routes. Results have shown that Ad distribution in peripheral blood, rectal tissue and in the lung is similar regardless of the route of administration. Rectal tissue is a principal target of HIV infection, and it is believed desirable that a vaccine vector replicate at this site in order to elicit local mucosal immunity. New Ad-recombinants are under development both for future clinical use and for pre-clinical studies in the rhesus macaque model. These include recombinants containing novel envelope inserts intended to generate broad, potent neutralizing antibodies. This work includes assessment of immunogenicity of the newly constructed recombinant vaccines in appropriate animal models. Overall, this project is focused on moving the replication-competent Ad-recombinant vaccine approach into phase I clinical trials. This is a novel approach, which has shown superior immunogenicity in non-human primate studies in comparison to non-replicating Ad-recombinant vaccines.